Anti-Proliferative Drugs
Anti-proliferative drugs, also known as anti-metabolites, anti-neoplastic agents and covalent DNA binding drugs, act by inhibiting essential metabolic pathways and are commonly used in the treatment of malignant diseases. However, their high toxicity to normal cells and severe side effects limit their use as therapeutic agents. Undesirable side effects include inter alfa anemia, emesis and balding due to cytotoxic effects on rapidly dividing normal cells, such as stem cells in the bone marrow, epithelial cells of the intestinal tract, hair follicle cells, etc.
Another major problem associated with anti-proliferative drugs is inherent or acquired resistance of tumors to the drugs. For example, although the initial remission rate following treatment with L-asparaginase is quite high in acute lymphoblastic leukemia (ALL) patients, relapse and associated drug resistance pose a significant clinical problem. Studies have demonstrated increased asparagine synthetase (AS) expression in asparaginase-resistant cells, which has led to the hypothesis that elevated AS activity permits drug-resistant survival of malignant cells (Aslanian, et al, 2001).
Drug Resistance
Multi-drug resistance (MDR), the resistance of cells to a broad spectrum of structurally unrelated cytotoxic drugs, is a severe problem in the clinic.
Many of the prevalent forms of human cancer resist effective chemotherapeutic intervention. Some tumor populations, especially adrenal, colon, jejunal, kidney and liver carcinomas, appear to have drug-resistant cells at the outset of treatment (Barrows, 1995). In other cases, a resistance-conferring genetic change occurs during treatment; the resistant daughter cells are able to proliferate in the environment of the drug. Whatever the cause, resistance often terminates the value of an anti-proliferative drug.
Clinical studies suggest that a common form of multidrug resistance in human cancers results from expression of the mdr1 gene that encodes P-glycoprotein, a plasma membrane, energy-dependent, multidrug efflux pump. The influx of chemotherapeutic agents into cells occurs mainly by passive diffusion across the cell membrane, driven by the drug's electrochemical-potential gradient. In MDR cells, P-glycoprotein actively pumps the drug out of the cells, reducing its intracellular concentration below lethal threshold.
MDR significantly limits the efficacy of many cancer chemotherapy regimens and is a major factor in their failure. MDR may account for intrinsic resistance in colorectal and renal cancer, and for acquired resistance observed in acute non-lymphocytic leukemia, malignant lymphomas, myeloma, and breast and ovarian carcinomas.
Efforts to counter MDR have primarily involved the use of hydrophobic competitors for P-glycoprotein binding. U.S. Pat. No. 6,605,638 discloses a method for inhibiting P-glycoprotein activity by contacting cells with branched fatty acid (BFAs) and their derivatives. Most of these competitors eventually fail to overcome MDR due to their interference with chemotherapeutic drug uptake and unexpected toxicities. As a consequence, anticipated benefits of these agents are often unattainable or unrealized.
Amino Acids and Proliferative Disease
Asparagine is an essential amino acid that is required by rapidly proliferating cells. Mammalian cells can synthesize asparagine from aspartate using the ATP-dependent enzyme asparagine synthetase (CE 6.3.5.4), which transfers the amino group from the amide of glutamine to the β-carboxyl of aspartate in a reaction that may be represented as: Glutamine+Aspartate+ATP+H2O=Glutamate+Asparagine+AMP+PPi.
Asparagine synthetase deficiency occurs in certain tumors, causing them to rely on an external supply of asparagine from other sources, such as serum. This observation led to the development of the enzyme L-asparaginase (type CE-2, CE 3.5.1.1) as a chemotherapeutic agent. L-asparaginase hydrolyzes L-asparagine to aspartate and ammonia, hence depleting L-asparagine from the serum and inhibiting tumor growth. L-asparaginase is used mainly in the treatment of Acute Lymphoblastic Leukemia (ALL) and shows some activity against other hematological cancers including acute non-lymphocytic leukemia (Whitecar, et al., 1970; Capizzi et al., 1970).
The L-asparaginase used in the clinic is available in two unmodified forms (native) purified from bacterial sources, and one as a PEGylated compound. U.S. Pat. No. 4,179,337 teaches PEGylated L-asparaginase, wherein the enzyme is coupled to PEG having a molecular weight of about 500 to 20,000 daltons.
The in vivo down-regulation of asparagine synthetase may provide an efficient mechanism for inhibiting tumor growth. However, cells respond to amino acid deprivation by a concerted increase in asparagine synthetase mRNA, protein, and enzymatic activity that involves transcriptional control of the asparagine synthetase gene. (Hutson, et al., 1997).
A metabolic approach was initially used to inhibit the activity of asparagine synthetase by the generation of L-asparagine and L-aspartic acid analogs. Analogs including 5-carboxamido-4-amino-3-isoxazolidone (Stammer et al., 1978) and N-substituted sulfonamides and N′-substituted sulfonylhydrazides have been prepared as sulfur analogues of L-asparagine (Brynes S et al., 1978a; Brynes S et al., 1978b). U.S. Pat. No. 4,348,522 teaches the salt of PALA, N-phosphonacetyl-L-aspartic acid, which has been shown to exhibit anti-tumor activity and is presently in clinical trials as combination chemotherapy for colorectal and pancreatic cancers (Whitehead et al, 2004a, 2004b).
Arginine has also been shown to be required for the growth of some tumor cell lines, including certain breast cancer cell lines (Caso, et al, 2004).
Other examples of amino acid derivatives and amino acid conjugates include sulphur containing tyrosine analogs having potent anti-melanoma activity (Thomas et al, 1999; McLaughlin et al, 1988; Tandon, et al, 1998) and antiproliferative activity (Purro et al, 2003). A proline analog of melphanan (Mel-pro) was shown to be a prodrug susceptible to the action of the cytosolic imidodipeptidase prolidase, suggesting that prolidase targeting may serve as a potential strategy in pharmacotherapy of breast cancer (Chrzanowski et al., 2003).
The use of prodrugs to impart desired characteristics such as increased bioavailability or increased site-specificity is a recognized concept in the art of pharmaceutical development. For example, direct or indirect conjugation of a drug to an antibody creates a stable conjugate that can arrive at the target site with minimum dissociation of the drug. Drug targeting may be combined with a mechanism of selective release of the drug for maximal potency.
The art neither teaches nor suggests compounds comprising a drug covalently linked to an amino acid via a side chain with a functional group selected from an amino group, a carboxyl, a sulfhydryl, a hydroxyl, a halogen, and a nitro group, useful for targeting drugs to neoplastic cells.
U.S. Pat. No. 4,296,105 describes doxorubicin derivatives linked to an optionally substituted amino acid at the hydroxy group of the amino acid residue, which possess in vitro a higher antitumor activity and lower toxicity than doxorubicin.
U.S. Pat. No. 5,962,216 teaches tumor activated prodrugs, which are unable to enter the cell, until cleaved by a factor or factors secreted by a target cell.
U.S. Pat. No. 5,650,386 teaches compositions comprising at least one active agent, and at least one modified non-alpha amino acid or poly amino acid, which acts as a carrier of the active agent. The amino acid modification includes acylation or sulfonation of at least one free amine group.
U.S. Pat. Nos. 6,623,731; 6,428,780 and 6,344,213 teach non-covalent mixtures comprising modified amino acids as carriers for biologically active agents.
U.S. Pat. No. 5,106,951 discloses a conjugate comprising an aromatic drug non-covalently intercalated between two aromatic side chains on an oligopeptide, and an antibody or antibody fragment covalently attached to the oligopeptide for targeting to cancer cells. U.S. Pat. No. 6,617,306 teaches a carrier for the in vivo delivery of a therapeutic agent, the carrier and therapeutic agent linked by a disulfide bond. In that patent, the carrier comprises a polymer, and at least one thiol compound conjugated to the polymer, such that the thiol group of the thiol compound and the thiol group of the therapeutic agent form a disulfide bond.
International patent application publication WO 00/33888 teaches cleavable anti-tumor and anti-inflammatory compounds comprising a therapeutic agent capable of entering a target cell, an oligopeptide, a stabilizing group and an optional linker.
It is to be explicitly understood that the present invention excludes known covalently linked conjugates of therapeutic agents and diagnostic agents to amino acid residues, enzymes, growth factors, peptide ligands of receptors, antibodies, as exemplified for instance in U.S. Pat. Nos. 5,106,951 and 4,401,592, among others.
There remains an unmet medical need for compounds and compositions capable of overcoming multi-drug resistance in tumors and of targeting tumors while obviating cytotoxic damage to normal tissues.